US5801039A - Enzymes for detergents - Google Patents

Enzymes for detergents Download PDF

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US5801039A
US5801039A US08/566,369 US56636995A US5801039A US 5801039 A US5801039 A US 5801039A US 56636995 A US56636995 A US 56636995A US 5801039 A US5801039 A US 5801039A
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amino acid
protease
blap
substitution
group
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Karl-Heinz Maurer
Albrecht Weiss
Christian G. Paech
Dean W. Goddette
Teresa M. Christianson
Maria R. Tang
Charles Ronald Wilson
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Cognis Gesellschaft fuer Bio und Umwelt Technologie mbH
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Cognis Gesellschaft fuer Bio und Umwelt Technologie mbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase

Definitions

  • This invention relates to novel mutant proteolytic enzymes with improved properties relative to the wild type enzyme in cleaning and detergent formulations, to nucleotide sequences encoding the improved proteases, and to host organisms containing the nucleotide sequences encoding the novel proteases.
  • This invention also includes within its scope new and improved detergent and cleansing compositions containing an effective cleansing amount of said enzymes.
  • Subtilisins are a family of bacterial extracellular proteases with molecular masses of 20,000 to 45,000 daltons produced by a soil bacillus e.g. Bacillus amyloliquefaciens.
  • Proteases are enzymes which catalyze the hydrolysis of peptide linkages in protein and peptide substrates and of ester bonds in some terminal esters.
  • Subtilisins belong to the group of serine proteases which initiate the nucleophilic attack on the peptide (ester) bond by a serine residue at the active site.
  • Subtilisins are physically and chemically well characterized enzymes.
  • subtilisins The three-dimensional structure of several subtilisins has been elucidated in detail by X-ray diffraction studies (Betzel, C., Pal, G. P., and Saenger, W. (1988) Eur. J. Biochem. 178, 155-171; Bott, R., Ultsch, M., Kossiakoff, A., Graycar, T., Katz, B., and Power, S. (1988) J. Biol. Chem. 263, 7895-7906; Goddette, D. W., Paech, C., Yang, S. S., Mielenz, J. R., Bystroff, C., Wilke, M., and Fletterick, R. J. (1992) J. Mol.
  • subtilisins are widely used in commercial products (for example, in laundry and dish washing detergents, contact lens cleaners) and for research purposes (catalysts in synthetic organic chemistry).
  • One member of the subtilisin family a highly alkaline protease for use in detergent formulations has been described in patent application WO 91/02792.
  • This Bacillus lentus alkaline protease (BLAP) can be obtained in commercial quantities from Bacillus licheniformis ATCC 53926 strain transformed by an expression plasmid harboring the wild type BLAP gene under the control of the B. licheniformis ATCC 53926 alkaline protease gene promoter.
  • the crystal structure of BLAP has been deduced (Goddette, D.
  • EP 0260105 teaches the construction of subtilisin BPN' mutants with altered transesterification rate/hydrolysis rate ratios and nucleophile specificities by changing specific amino acid residues within 15 ⁇ of the catalytic triad.
  • Russell, A. J., and Fersht, A. R. (1987) J. Mol. Biol. 193: 803-813 teach the isolation of a subtilisin BPN' mutant (DO99S) that had a change in the surface charge 14 to 15 ⁇ from the active site. This substitution causes an effect on the pH dependence of the subtilisin's catalytic reaction.
  • EP 0130756, EP 0247647, and U.S. Pat. No. 4,760,025 teach a saturation mutation method where one or multiple mutations are introduced into the subtilisin BPN' at amino acid residues (BPN' numbering) Asp32, Asn155, Tyr104, Met222, Gly166, His64, Ser221, Gly169, Glul156, Ser33, Phe189, Tyr217, and/or Ala152. Using this approach mutant proteases exhibiting improved oxidative stability, altered substrate specificity, and/or altered pH activity are obtained. These publications also teach that mutations within the active site region of the protease are the most likely to influence activity. However, neither EP0130756, EP 0247647, nor U.S. Pat. No. 4,760,025 teach a method for predicting amino acid alterations that will improve the wash performance of the protease.
  • subtilisins Most of the information on the catalytic activity of subtilisins has been collected by examining the hydrolysis of small, well defined peptide substrates. Yet, little is known about interactions with large protein substrates. This is especially true for the wash performance of proteases where the substrate is attached to a textile surface and catalysis takes place in presence of interfering compounds such as bleach, tensides, and builders.
  • EP 0328229 teaches the isolation and characterization of PB92 subtilisin mutants with improved properties for laundry detergent applications based upon wash test results. It teaches that biochemical properties are not reliable parameters for predicting enzyme performance in the wash. Methods for selection of mutations involve the substitution of amino acids by other amino acids in the same category (polar, nonpolar, aromatic, charged, aliphatic, and neutral), the substitution of polar amino acids asparagine and glutamine by charged amino acids, and increasing the anionic character of the protease at sites not involved with the active site. No method for identifying which specific amino acids should be altered is taught.
  • Patent application WO 91/00345 (Novo-Nordisk) teaches a method to improve the wash performance of a subtilisin by the modification of the isoelectric point of subtilisin Carlsberg and subtilisin 309 to match the pH of the washing solution, where the enzyme is supposed to be used.
  • WO 91/00345 also teaches that changes in amino acids more than 15 ⁇ from the catalytic triad can result in changes in the kinetic properties of the enzyme.
  • a total of 116 different amino acids out of a total of 269 amino acids present in subtilisin 309 are suggested as possible sites for substitution, addition or deletion to modify the net electric charge of the enzyme.
  • Patent application WO 92/11357 (Novo-Nordisk) teaches increased pH-stability and improved washability of subtilisins by reduction of pH-dependent charges of the molecule. This means the introduction of mutations to approach substantial constancy of charge over a pH range. Preferably, an almost zero net charge change in the pH range from 7 to 11.
  • European patent application No. 0 57 049 A1 discloses certain mutant proteolytic enzymes. These enzymes are said to have at least 70% homology with the amino acid sequence of PB92 serine protease and differ by at least one amino acid corresponding to 99, 102, 116, 126, 127, 128, 130, 160, 203, 211, and 212 in the PB92 sering protease.
  • the mutant protease is prepared by growing a microorganism host strain transformed with an expression vector comprising a DNA sequence and encoding a mutant protease to produce the designed mutant protease.
  • the wild-type protease from which the mutant proteases according to the invention are derived is a Bacillus lentus alkaline protease (BLAP) obtained from DSM 5483 having 269 amino acid residues, a molecular mass of 26,823 daltons and a calculated isoelectric point of 9.7 based on standard pK values.
  • the BLAP gene is obtained by isolating the chromosomal DNA from the B. lentus strain DSM 5483, constructing DNA probes having homology to putative DNA sequences encoding regions of the B. lentus protease, preparing genomic libraries from the isolated chromosomal DNA and screening the libraries for the gene of interest by hybridization to the probes.
  • Mutants of B. lentus DSM 5483 protease with improved thermal and surfactant stability have been described in patent application Ser. No. 07/706,691 filed May 29, 1991 now U.S. Pat. No. 5,340,735 issued Aug. 23, 1994.
  • the mutations described in this invention are introduced into wild-type BLAP with the following amino acid replacements: S3T, V4I, A188P, V193M, and V199I (numbering according to the BLAP sequence).
  • FIG. 1 is a model of the substrate binding region of wild type BLAP with the synthetic substrate AAPF bound to the enzyme. The positions of arginine 99, serine 154 and leucine 217 in relation to the bound substrate are shown.
  • FIG. 2 shows the restriction map for Escherichia coli plasmid pCB13C which contains a hybrid gene fusion between the Bacillus licheniformis ATCC 53926 protease gene and the Bacillus lentus DSM 5483 BLAP gene.
  • the promoter, ribosomal binding site and presequence (53926) from ATCC 53926 were fused to the pro and mature sequence of the BLAP gene.
  • the transcription terminator of ATCC 53926 (T-53926) was appended to the BLAP coding sequence.
  • FIG. 3 shows the DNA sequence for the AvaI/ClaI fragment from the N-terminal region of the ATCC 53926 alkaline protease gene discussed in Example 2.
  • the fragment includes the putative promoter, ribosomal binding site, initiation codon, and most of the pre sequence.
  • the 292 base pair fragment is flanked by AvaI and ClaI restriction sites at its 5' and 3' ends, respectively.
  • FIG. 4 shows the restriction map for E. coli plasmid pMc13C which is derived from pMac5-8 and contains the BLAP gene.
  • the ampicillin resisitance (Ap R ) gene present in pMc13C carries an amber mutation which renders it inactive.
  • the plasmid still encodes resistance to chloramphenicol (Cm R ).
  • FIG. 5 shows the restriction map for Bacillus plasmid pCB76M131 which contains the gene encoding BLAP variant M131. Included is the hybrid fusion between the ATCC 53926 protease and BLAP as described for pCB13C in FIG. 2. Also presence is the transcription terminator sequence from the 53926 alkaline protease gene. This is a pUB110 based plasmid which encodes resistance to kanamycin.
  • FIG. 6 shows a restriction map for Bacillus plasmid pH70 which is a derivative of plasmid pUB110 containing the ATCC 53926 alkaline protease gene.
  • An EcoRI/BamHI fragment carrying the protease gene was cloned between the EcoRI and BamHI sites on pUB110. This plasmid is discussed in Example 3: Cloning of mutant protease genes. Plasmid pH70 encodes resistance to kanamycin.
  • FIG. 7 shows a restriction map for Bacillus plasmid pC51 which is a derivative of plasmid pBC16 carrying the ATCC 53926 alkaline protease gene.
  • An EcoRI/BamHI carrying the protease gene was cloned between an EcoRI site and the BamHI sites on plasmid pBC16. This plasmid is discussed in Example 3: Cloning of mutant protease genes.
  • Plasmid pC51 encodes resistance to tetracycline.
  • FIG. 8 shows a restriction map for Bacillus plasmid pBC56M131 which encodes the gene for BLAP variant M131. Included is the hybrid fusion between the ATCC 53926 alkaline protease gene and the BLAP gene as described in FIG. 2 for pCB13C. Also present is the transcription terminator sequence from the ATCC 53926 alkaline protease gene. This is a pBC16 based plasmid which encodes resistance to tetracycline.
  • FIG. 9 shows the peptides produced by digestion of BLAP with trypsin.
  • BLAP 11 mg/ml -1
  • trypsin 1%, w/w, in 1 mM HCl
  • the sample was then acidified with 10% (v/v) Trifluroacetic acid (TCA) to a final concentration of 1% TCA.
  • TCA Trifluroacetic acid
  • the tryptic peptides correspond to the following BLAP sequences: Fragment #1-Ala1 to Arg10; Fragment #2-Val11 to Arg19; Fragment #3-Gly20 to Lys27; Fragment #4-Val28 to Arg44; Fragment #5-Gly45 to Lys92; Fragment #6-Val93 to Arg99; Fragment #7-Gly100 to Arg143; Fragment #8-Gly144 to Arg164; Fragment #9-Tyr165 to Arg180; Fragment #10-Ala181 to Lys229; Fragment #11-Gln230 to Lys231; Fragment #12-Asn232 to Arg241; Fragment #13-Asn242 to Lys245; and Fragment #14-Asn246 to Arg269.
  • Bacillus licheniformis E312 with plasmid pBC56M131 is deposited as ATCC 68614.
  • Escherichia coli WK6 with plasmid pMC13C is deposited as ATCC 68615.
  • E. coli GM33 with plasmid pCB13C is deposited as ATCC 68616.
  • E. coli WK6 with plasmid pMa5-8 is deposited as ATCC 68617.
  • E. coli WK6 with pMc5-8 is deposited as ATCC 68618.
  • One aspect of the present invention relates to a method for choosing amino acid alterations which can result in a mutant protease with improved wash performance.
  • Improved wash performance is obtained by introducing amino acid alterations within a region of the substrate binding pocket of the enzyme which results in an increased negative charge. According to the present invention, this can be achieved by increasing the number of negatively charged amino acids residues or decreasing the number of positively charged amino acid residue in the region of the substrate binding pocket of the protease within 7A of a bound substrate molecule such as AAPF.
  • amino acid alterations at positions 99, 154 and 211 within Bacillus lentus Alkaline Protease (BLAP) variants M130 and M131 were shown to enhance the wash performance of the enzyme.
  • a second aspect of the present invention relates to mutant proteolytic enzymes which have an improved wash performance relative to the wild type protease as determined by laboratory tests.
  • the mutations described in this invention are introduced into BLAP variants M130 or M131 which have been previously described in patent application Ser. No. 07/706,691 filed May 29, 1991 now U.S. Pat. No. 5,340,735 issued Aug. 23, 1994. Both M130 and M131 have been shown to have improved stability as compared to the wild type protease.
  • Mutant M130 contains four amino acid alterations: S3T; A188P/V193M and V199I.
  • Mutant M131 contains five amino acid alterations: S3T; V41; A188P; V193M and V199I.
  • the system used to designate preferred proteases first list the amino acid residue in the mature form of BLAP at the numbered position followed by the replacement amino acid using the accepted one letter amino acid codes.
  • the amino acid sequences for proteases M130 and M131 are given in SEQ ID NO: 2 and SEQ ID NO: 1, respectively, contained in patent application Ser. No. 07/706,691 filed May 29, 1991 now U.S. Pat. No. 5,340,735 issued Aug. 23, 1994.
  • the Bacillus lentus DSM 5483 BLAP DNA and protein sequences are shown herein in SEQ ID No: 19 and SEQ ID No: 22, respectively.
  • the M130 DNA and protein sequences are shown herein in SEQ ID No: 20 and SEQ ID No: 23, respectively.
  • the M131 DNA and protein sequences are shown herein in SEQ ID No: 21 and SEQ ID No: 24, respectively. Both M130 and M131 served as the basis for additional amino acid alterations to achieve proteases with improved wash performance.
  • the mutant proteases according to the invention are those derived by the replacement of at least one amino acid residue of mutant proteases M130 or M131 wherein said amino acid residue is selected from the group consisting of arginine at position 99, serine at position 154 and leucine at position 211.
  • Table 2 provides a description of the BLAP mutant proteases claimed in the invention which include F11, F43, F44, F45, F46, F47, F49, F54 and F55.
  • F11 shown in Table 2 is derived from M130 and contains an arginine at position 99 replaced by a serine (R99S) along with the other mutations present in M130 which include: a serine at position 3 replaced by a threonine (S3T); an alanine at position 188 replaced by a proline (A188P); a valine at position 193 replaced by a methionine (V193M) and a valine at position 199 replaced by a isoleucine (V1991).
  • F43 through F49 are derived from M131.
  • Mutants F54 and F55 are derived from mutant F49.
  • the amino acid sequences of the preferred proteolytic enzymes F11, F43, F44, F45, F46, F47, F49, F54 and F55 are given in SEQ ID NO: 1 to SEQ ID NO: 9, respectively, of this application.
  • a third aspect of this invention relates to the genes which encode the mutant proteases listed in Table 2.
  • the genetic construction of all mutant proteases in this invention are described in detail in Example 2. In all cases the mutations introducing the amino acid alterations are constructed using known procedures.
  • Each hybrid gene encoding one of the mutant proteases listed above is comprised in the direction of transcription, a promoter, a ribosomal binding site, and initiation codon and the major portion of the pre region of the B.
  • licheniformis ATCC 53926 alkaline protease gene operably linked to a portion of the pre region and all of the pro and mature regions of a variant of the BLAP gene followed by the transcription terminator sequence for the alkaline protease gene from ATCC 53926.
  • the hybrid gene may be integrated into the chromosome of the host or is carried on a plasmid which replicates within the Bacillus strain of choice.
  • plasmid pUB110 or a derivative of pUB110 is the plasmid of choice for protease production in strains of Bacillus subtilis
  • plasmid pBC16 or a derivative of pBC16 is the choice for protease production in strains of B. licheniformis.
  • the mutant proteases are produced by growing the Bacillus strains transformed by plasmids containing the hybrid genes in a suitable medium.
  • washability relates to the number and distribution of charged amino acid residues in the substrate binding region.
  • An improvement in the wash performance was seen with an increased number of negatively charged amino acid residues or with a decrease of the number of positively charged amino acid residues in the substrate binding region. Accordingly, the BLAP mutants claimed herein have a reduced positive net charge in the substrate binding region and they show improved washability.
  • FIG. 1 shows the characteristic structure of the substrate binding pocket of wild type BLAP with the synthetic substrate AAPF.
  • AAPF bound to the enzyme was modeled on crystallographic data of subtilisin-inhibitor complexes found in the literature and available from the Brookhaven Protein Data Bank.
  • BLAP variants M130 and M131 within the substrate binding pocket is essentially identical to wild type BLAP based upon Xray crystallography.
  • the structure of wild type BLAP to a 1.4 ⁇ resolution has been published in patent application Ser. No. 07/706,691 filed May 29, 1991 now U.S. Pat. No. 5,340,735 issued Aug. 23, 1994 and the corresponding atomic coordinates deposited with the Brookhaven Protein Data Bank.
  • amino acid alterations at positions 99, 154 and 211 within BLAP variants M130 and M131 were shown to enhance the wash performance of the enzyme.
  • the parental amino acids occupying these sites, Arginine 99, Serine 154, and Leucine 211 are depicted in FIG. 1. All three amino acids are located within the specified 7 ⁇ radius of AAPF.
  • Genes which express the mutant B. lentus DSM 5483 proteases according to the invention are made by altering one or more codons of the wild-type B. lentus DSM 5483 alkaline protease gene.
  • Protease M130 was derived from BLAP by introducing mutations S3T, A188P, V193M and V199I.
  • Protease M131 was derived from BLAP by introducing the mutations S3T, V4I, A188P, V193M, and V199I.
  • Genes encoding proteases M130 and M131 were constructed using the pMac procedure (Stanssens, P., Opsomer, C., McKeown, Y.
  • Proteases M130 and M131 have been previously described in patent application Ser. No. 07/706,691 filed May 29, 1991 now U.S. Pat. No. 5,340,735 issued Aug. 23, 1994. Proteases M130 and M131 exhibit improved thermal and surfactant stability over the wild type BLAP and served as the basis for developing proteases with improved wash performance.
  • the genetic techniques used to modify the BLAP gene to produce the M130 and M131 proteases have also been described in patent application WO 91/02792. The description in WO 91/02792 incorporated herein by reference for purposes of indicating the state of the art.
  • Proteases M130 and M131 were derived from the B. lentus DSM 5483 alkaline protease (BLAP) by site-specific mutagenesis of DNA encoding the mature form of wild type BLAP.
  • the DNA fragment encoding the mature form of wild type BLAP was prepared using plasmid pCB13C (FIG. 2).
  • Plasmid pCB13C contains a hybrid fusion between the B. licheniformis ATCC 53926 protease gene and the B.
  • this hybrid fusion contains DNA encoding the promoter, ribosomal binding site, and 21 residues of the pre sequence from the ATCC 53926 protease gene fused to a DNA sequence encoding the last five residues of the BLAP pre sequence and all of the pro and mature residues of BLAP.
  • This fusion is referred to as the ClaI fusion because this restriction site is located at the juncture between the ATCC 53926 and DSM 5483 DNA's.
  • a new ClaI restriction site had to be introduced into the ATCC 53926 alkaline protease gene near to the junction of the pre and pro sequences.
  • the ClaI site was introduced into the ATCC 53926 alkaline protease gene by using the polymerase chain reaction (PCR) to amplify a DNA fragment containing sequence information from the N-terminal part of the ATCC 53926 alkaline protease gene.
  • the amplified fragment included the ATCC 53926 alkaline protease promoter, ribosomal binding site, initiation codon, and most of the pre sequence.
  • the DNA sequence of this fragment is shown in FIG. 3. This 292 bp DNA fragment was flanked by AvaI and ClaI restriction sites at its 5' and 3' ends, respectively.
  • the BLAP gene already contained a naturally occurring ClaI site at the corresponding position. Analysis of the DNA sequence across the fusion of the ATCC 53926 and BLAP genes confirmed the expected DNA and amino acid sequences.
  • the BLAP gene is subcloned into the mutagenesis vector pMa5-8 . This is accomplished by synthesizing a DNA fragment containing the ClaI fusion gene and the ATCC 53926 transcription terminator as a SalI cassette using the PCR. The PCR was carried out using conditions as described by the manufacturer (Perkin Elmer Cetus, Norwalk, Conn.). In the PCR, two synthetic oligonucleotides bearing SalI sites are used as primers and Escherichia coil vector pCB13C DNA as a template.
  • Chloramphenicol resistant (Cm R ) transformants are screened for the presence of an insert and a correct plasmid construct pMc13C is identified as shown in FIG. 4.
  • the mutation(s) is introduced using synthetic DNA oligonucleotides according to a modification of a published protocol (Stanssens, P., et. al. (1989) Nucleic Acids Res. 17, 4441-4454).
  • the oligonucleotide containing the mutation(s) to be introduced is annealed to a gapped duplex (gd) structure which carries the BLAP gene on a segment of single stranded (ss) DNA.
  • the gapped duplex can be formed by annealing linear ss DNA from pMc13C with denatured and restricted pMa5-8 DNA.
  • Plasmid pMa5-8 contains an active ampicillin resistance gene but has an inactivating point mutation in the chloramphenicol resistance gene
  • plasmid pMc13C contains, in addition to an intact BLAP gene, an active chloramphenicol resistance gene, but has an inactivating point mutation in the ampicillin resistance gene.
  • the annealed product is the gd DNA which is a double stranded heteroduplex with a ss DNA gap spanning the entire cloned BLAP gene.
  • the mutant oligonucleotide is able to anneal to homologous ss BLAP DNA within the gap and the remaining gap is filled in by DNA polymerase I (Klenow fragment) and ligated using T4 DNA ligase (New England Biolabs Inc., Beverly, Mass. NEB!).
  • the mutagenic efficiency of such a system can be improved by the use of Exonuclease III (Exo III, NEB).
  • Exo III is an exodeoxyribonuclease that digests double stranded DNA from the 3' end.
  • the pMa mutant derivative of the first mutagenesis round can be used for a second round of mutagenesis by preparing ss DNA of that species and annealing it to XbaI/HindIII restricted and denatured DNA of pMc5-8.
  • Plasmid pMc5-8 is identical to pMa5-8 except that it contains an active chloramphenicol resistance gene and an inactive ampicillin resistance gene.
  • the general procedure is the same as that described above.
  • the construction of the genes encoding proteases M130 and M131 required two rounds of mutagenesis.
  • an oligonucleotide was designed to introduce mutations A188P, V193M, and V199I.
  • an oligonucleotide was designed to introduce mutation S3T in the case of M130 and mutations S3T and V4I in the case of M131. The presence of all of these mutations was verified by DNA sequencing.
  • Mutations R99G, R99A, R99S, S154D, S154E and L211D were introduced into the gene encoding M131 protease using PCR mutagenesis by overlap extension. Mutation R99S was introduced into the gene encoding protease M130 using PCR mutagenesis by overlap extension.
  • Construct pCB76M131 (FIG. 5) was used to construct mutants F43, F44, F45, F46, F47, and F49 (Table 2).
  • Construct pCB76M13O was used to construct mutant F11 and construct pCB76F49 (a newly constructed mutant) was used to construct mutants F54 and F55.
  • a DNA Thermal Cycler Perkin Elmer Cetus
  • GeneAmp kit Perkin Elmer Cetus
  • AmpliWax Perkin Elmer Cetus
  • 0.5 ml sterile polypropylene tubes Perkin Elmer Cetus
  • Microcon-100 concentrators Amicon, Beverly, MA
  • TE buffer (10 mM tris-(hydroxymethyl)aminomethane Tris!, 1 mM disodium ethylenediamine tetraacetic acid (EDTA), adjusted to pH 8 with 2N HCl
  • Minigel electrophoresis apparatus Hoefer Scientific, San Francisco, Calif.
  • 1% (w/v) SeaKem agarose gel FMC, Rockland, Me.
  • TBE buffer 0.089M Tris, 0.089M boric acid, 2 mM EDTA
  • PCR's were carried out using the GeneAmp kit and Ampliwax as specified by the manufacturer. They were subjected to 1 cycle of denaturation (3 minutes, 95° C.), annealing (2 minutes, 50° C.) and extension (2 minutes, 72° C.) and 30 cycles of denaturation (1 minute, 94° C.), annealing (1 minutes, 50° C.) and extension (1 minute, 72° C.) using a DNA Thermal Cycler. Each cycle was extended for 10 sec at 72° C.
  • telomeres The pUB110 forms of these plasmids were chosen because they provide higher yields of protease in the B. subtilis DB104 host than the pBC16 forms of the plasmids.
  • PCR fragments were checked by agarose gel electrophoresis and cleaned using a Microcon-100 concentrator (Amicon) after each round of PCR.
  • the fragments were digested with either NheI/XbaI or NheI/SstI (depending on the location of the intended mutation) and cloned back into pCB76M131 DNA in the case of mutants F43, F44, F45, F46, F47 and F49, pCB76Ml3O DNA in the case of mutant F11 and pCB76F49 in the case of mutants F54 and F55 using B. subtilis DB104 competent cells as previously described. Plasmid DNA isolation was accomplished using ion exchange minicolumns (QIAGEN, Inc., Chatsworth, Calif.) and mutations were checked by Sanger ds DNA sequencing as previously described.
  • Proteases M130 and M131 can be produced by transferring the respective gene encoding either M130 or M131 from the particular E. coli pMc13C derivative vector into a plasmid vector which can replicate in Bacillus. To accomplish this, the desired mutant gene is separated from the appropriate pMc13C plasmid by digestion with the restriction endonucleases AvaI and SstI, followed by ligation to the larger AvaI/SstI fragment from either plasmid pH70 (FIG. 6) or pC51 (FIG. 7).
  • AvaI/SstI fragments from pH70 and pC51 include the DNA sequences necessary for replication in Bacillus and encode either kanamycin resistance (Km R ) or tetracycline resistance (Tc R ), respectively.
  • Plasmid pH70 is constructed by cloning the ATCC 53926 alkaline protease gene carried on a EcoRI/BamHI DNA fragment into the Km R plasmid pUB110 between the EcoRI and BamHI sites.
  • Plasmid pC51 is constructed by cloning the ATCC 53926 protease gene carried on a EcoRI/BamHI fragment into the Tc R plasmid pBC16 between the EcoRI and BamHI sites.
  • the larger AvaI/SstI fragment from either pH70 or pC51 used for cloning the DNA fragment encoding either M130 or M131 is first purified from other plasmid DNA fragments by high pressure liquid chromatography (HPLC) on an anion exchange column (Gen-Pak FAX, 4.6 mm diameter, 100 mm long; Waters, Milford, Mass.). Conditions for elution of the DNA are a flow rate of 0.75 ml.
  • Buffer A 25 mM Tris, containing 1 mM EDTA and adjusted to pH 8.0 with 2N HCl
  • Buffer B 25 mM Tris, containing 1 mM EDTA, 1M NaCl, and adjusted to pH 8.0 with 2N HCl
  • the two ligated DNA's are transformed into B. subtilis DB104.
  • the genes encoding the major alkaline and neutral proteases present in this strain have been inactivated (Kawamura, F. and Doi, R. A. (1984) J. Bacteriol. 160, 442-444).
  • Cells of B. subtilis DB104 transformed by these plasmids grow on a nutrient-skim milk agar in the presence of either kanamycin or tetracycline.
  • Transformants of DB104 that manufacture mutant protease are identified by the formation of clear zones of hydrolysis in the skim milk.
  • Confirmation that the protease-producing transformants carry a plasmid-borne M130 or M131 gene with the desired mutation(s) is accomplished by purifying plasmid DNA from a culture of each transformant.
  • the plasmid DNA is purified away from cell protein and chromosomal DNA by SDS-salt precipitation followed by chromatography over a QIAGEN ion-exchange column (QIAGEN,Inc., Chatsworth, Calif.).
  • AvaI/SstI digested plasmid DNAs from different transformants are compared with AvaI/SstI-digested derivatives of plasmid pH70 or pC51 known to carry an intact BLAP gene.
  • Restriction digests of these plasmids are compared by agarose gel electrophoresis to identify plasmids that have the proper-sized AvaI/SstI DNA fragments. Selected plasmid DNAs are then sequenced across the region of the expected M130 or M131 mutations to confirm that the desired mutation(s) are present.
  • Genes M130 and M131 cloned into the derivative of plasmid pC51(TcR) are designated plasmids pCB56M130 and pCB56M131 respectively (FIG. 8), while the same genes cloned into a derivative of plasmid pH70 are designated plasmids pCB76M130 and pCB76M131 respectively (FIG. 5).
  • One or more clones of each BLAP mutation are stored frozen in 15% glycerol at -70° C. and also cultivated in shake flasks to produce mutant protease for characterization.
  • the amplified PCR fragments are cloned back into plasmids pCB76M130, pCB76M131 or pCB76F49.
  • the AvaI/SstI fragment carrying the modified M130, M131 or F49 genes is cloned back into the pC51 type vector as described previously.
  • Wild type BLAP protein and mutant proteins were produced by transformed Bacillus subtilis DB 104 in shake flasks. A hot loop was used to streak each mutant strain from a frozen cryovial culture onto an LB-skim milk agar containing either 20 ⁇ g ⁇ ml -1 of kanamycin or 15 ⁇ g ⁇ ml -1 of tetracycline. The plates were incubated at 37° C. for 20 to 24 hours.
  • a single, isolated colony producing a good zone of hydrolysis of the skim milk was picked into a 250 ml Erlenmeyer flask containing about 50 ml Luria Broth (LB) which contained either 20 ⁇ g ⁇ ml -1 kanamycin or 15 ⁇ g ⁇ ml -1 of tetracycline.
  • the broth was incubated in a New Brunswick Series 25 Incubator Shaker at 37° C. with shaking at 280 rpm for 7 to 8 hours.
  • Either 2.5 ml of the turbid preculture was transferred into 50 ml of MLBSP containing either 20 ⁇ g ⁇ ml -1 kanamycin or 15 ⁇ g ⁇ ml -1 of tetracycline in each of four baffled 500 ml flasks, or 5 ml of preculture was used as an inoculum for 100 ml of MLBSP broth with antibiotic contained in each of two 500 ml baffled flasks (a 5%, v/v, transfer). All flasks were incubated at 240 rpm and 37° C. for 64 hours.
  • the concentrate was dialyzed for 16 hours against 20 mM sodium phosphate, pH 7.0 ( ⁇ phosphate buffer ⁇ ), or against 20 mM N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), containing 1 mM CaCl 2 and adjusted to pH 7.8 with NaOH ( ⁇ HEPES buffer ⁇ ).
  • the dialysate was clarified by centrifugation (20,000 ⁇ g av . for 10 minutes) and the pH of the solution, if necessary, was re-adjusted.
  • the enzyme purified in phosphate buffer was concentrated and desalted by ultrafiltration using Centricon tubes (molecular-weight-cut-off 10,000; Amicon).
  • the protein concentration was determined by the bicinchoninic acid method (BCA method, Pierce Chemical Co., Rockford, Ill.).
  • BCA method Pierce Chemical Co., Rockford, Ill.
  • the pooled fractions with enzyme purified in HEPES buffer were mixed with a 5 to 8-fold volume excess of acetone at -20° C.
  • the protein was allowed to precipitate for 4 minutes, and the mixture was centrifuged for 4 minutes at 6,600 ⁇ g av .. The supernatant was discarded, the pellet was briefly exposed to vacuum (water aspirator) to remove most of the acetone, and the pellet was dissolved in 20 mM 2-(N-morpholino)ethanesulfonic acid (MES), containing 1 mM CaCl 2 and adjusted to pH 5.8 with 2N NaOH, to give an approximate protein concentration of 30 mg ⁇ ml -1 .
  • MES 2-(N-morpholino)ethanesulfonic acid
  • the solution was clarified by centrifugation in an Eppendorf centrifuge for 3 minutes at full speed (13,000 ⁇ g max .) and stored frozen until used.
  • the protein concentration was determined by the biuret method (Gornall, A. G., Bardawill, C. S., and David, M. M. (1948) J. Biol. Chem. 177, 751-766).
  • the active enzyme concentration was determined by active site titration with the inhibitor phenylmethyl sulfonylfluoride.
  • a protease solution was prepared in 10 mM sodium phosphate, pH 6.5, at an approximate concentration of 100 ⁇ M based on protein determination.
  • Inhibitor concentrations were equivalent to an estimated molar ratio of 0.25, 0.5, 0.75, 1.0 and 1.25. The mixtures were allowed to react for one hour at room temperature. Residual enzyme activity was measured spectrophotometrically by the AAPF-PNA method (see below).
  • protease assays Two different protease assays were used. With the HPE method protease activity was established at a single concentration of casein as substrate. In the AAPF-PNA assay initial rates of succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanyl-p-nitroanilide (AAPF-pNA; Bachem Bioscience, Philadelphia, Pa.) supported catalysis were used to determine the kinetic parameters K m , k cat , and k cat /K m .
  • AAPF-pNA succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanyl-p-nitroanilide
  • Proteolytic activity was determined by a discontinuous assay using casein as a substrate.
  • the final concentrations of the substrate solution were 12 mg ⁇ ml -1 of casein (prepared according to Hammarsten; Merck, Darmstadt, #2242) and 30 mM Tris in synthetic tap water.
  • Synthetic tap water is a solution of 0.029% (w/v) CaCl 2 ⁇ 2H 2 O, 0.014% (w/v) MgCl 2 ⁇ 6H 2 O, and 0.021% (w/v) NaHCO 3 with a hardness of 15° dH (yer Harte, German hardness).
  • the substrate solution is heated to 70° C. and pH is adjusted to 8.5 at 50° C. using 0.1N NaOH.
  • the protease solution is prepared with 2% (w/v) anhydrous pentasodium tripolyphosphate in synthetic tap water, adjusted to pH 8.5 with hydrochloric acid.
  • TCA trichloroacetic acid
  • TCA-insoluble protein After cooling on ice for 15 minutes the TCA-insoluble protein is removed by centrifugation, an aliquot of 900 ⁇ l is mixed with 300 ⁇ l of 2N NaOH and the absorbance of this mixture containing TCA-soluble peptides is recorded at 290 nm. Control values are produced by adding 600 ⁇ l of TCA solution to 600 ⁇ l of casein solution followed by 200 ⁇ l of enzyme solution.
  • Protease samples were diluted with 50% (v/v) 1,2-propanediol in 100 mM Tris, adjusted with 2N HCl to pH 8.6 at 25° C. ( ⁇ Tris-propanediol buffer ⁇ ), in which they were stable for at least 6 h at room temperature.
  • a stock solution of 160 mM AAPF-pNA was prepared in dimethylsulfoxide dried over molecular sieve beads (Aldrich; 4 ⁇ , 4-8 mesh) for at least 24 h prior to use. Fixed point assays were performed at 25° C.
  • Kinetic parameters were calculated from a velocity vs. substrate concentration plot constructed from initial rates measured once each at 12 different AAPF-pNA concentrations ranging from 0.16 to 3.2 mM. Data were fitted to a hyperbolic curve and proportionally weighted using the program ENZFITTER (Leatherbarrow, R. J. (1987) ENZFITTER. Biosoft, Cambridge, UK). A nominal molecular weight of 26.8 kDa was used in all calculations that required the interconversion of protein concentration and molarity of protease enzyme (Table 4).
  • the HPLC equipment was from Waters and consisted of an autosampler (model 715 Ultra Wisp), a dual pump system (model 600E) and a diode array detector (model 990). Sampling and gradient formation was governed by Waters' software program ⁇ 990 + Powerline ⁇ . Tryptic peptides were separated on a C 18 column (Vydac model 218TP54; 4.6 ⁇ 250 mm; 5 ⁇ particle size; 300 ⁇ pore size). In line with the separation column was a C 18 guard column (Vydac model 218FSK104, 10 ⁇ particle size). Separation column and guard column were housed in a column heater set to 30° ⁇ 1° C.
  • Stability of the protease mutants to tensides was tested with SDS as typical anionic detergent. Stability was tested in 50 mM sodium carbonate, pH 10.5 at 50° C., containing 1% (w/v) SDS. Protease proteins were incubated at a final protein concentration of 0.25 mg ⁇ ml -1 . Periodically, an aliquot was removed from the incubation mixture and diluted into Tris-propanediol buffer chilled on ice. Residual protease activity was determined by the AAPF-pNA assay at a substrate concentration of 1.1 mM. Stability is expressed as half-life (t 1/2 ) of activity determined from semi-logarithmic plots of residual activity as function of time.
  • the wash performance was tested in a specially developed washing test using cotton swatches soiled with egg and soot (ER) and with blood, milk and soot (BMR).
  • the washing tests were performed in an Atlas launderometer (type LP 2), equipped with stainless steel test vessels each containing a defined detergent composition plus the protease to be tested.
  • the pre-washed cotton was soiled with a defined amount of soil and air-dried for 6 days.
  • the launderometer beakers were filled with 1 swatch of soiled and 3 swatches of unsoiled cotton.
  • Ten metal balls (10 mm diameter) were added for mechanical treatment.
  • the washing time was 30 minutes with a final temperature of 30° C. reached after 4 minutes of heating.
  • Laundry detergents for these tests were of typical composition for European usage (Jakobi, G. and Lohr, A. (1987) Detergents and Textile Washing, VCH, Weinheim, Germany).
  • concentrations of a delicate fabric detergent for easy-care and colored fabrics (5-15% (w/w) anionic surfactants, 1-5% (w/w) nonionic surfactants), a heavy duty compact detergent (8% (w/w) anionic surfactants, 6% (w/w) nonionic surfactants, with tetraacetylethylenediamine (TAED) and perborate bleach) and a super compact detergent concentrate (18% (w/w) anionic surfactants, 2.5% (w/w) nonionic surfactants, with TAED and perborate bleach) were 0.5 g, 0.5 g, and 0.4 g, respectively, in 100 ml of water at 16°dH (yer Harte, German hardness).
  • the super compact detergent is an extruded detergent granulate, described by a number of patent applications (WO 91/02047, WO 91/13678, WO 93/02176, WO 93/15180, WO 94/01526). In all cases the pH was 10.4. A protease was added to washing solutions on the basis of its enzymatic activity measured in HPE at a ratio of 0, 50, 100, 200, 300, 400, 500, 700 and 1000 HPE per gram of detergent.
  • the improvement in washing performance was determined by the ratio of wild type enzyme necessary to achieve a standard ⁇ Rem, versus the amount of mutant enzyme to achieve an identical effect. Thus an improvement of 2 indicates that half of the mutant enzyme is needed to get the same effect as with the wild type enzyme.
  • An enzymatic detergent composition is prepared by mixing about 30 wt % of a concentrated preparation of a subtilisin protease mutant comprising one or more of the following mutations: R99G, R99A, R99S, R99E, L211D, L211E, S154D and S154E, with about 5 wt % of cellulose, 5 wt % of saccharose, 20 wt % of wheat flour, 30 wt % of starch, 5 wt % of carboxymethylcellulose and 5% of polyethylene glycol (mw 20,000).
  • the resulting mixture is granulated by extrusion granulation. After drying, the granulate has an activity of 70,000 to 250,000 HPE/g.
  • the granulate is coated with polyethylene glycol (mw 6000) containing TiO 2 .
  • the granulated protease enzyme is then mixed with 100 g Heavy Duty Compact Detergent (Porsil supra) containing sodium perborate ⁇ TAED resulting in a standard protease activity of 1200 HPE/g.
  • An enzymatic detergent composition is prepared according to Example 11 except the protease is replaced by a mutated Bacillus lentus DSM 5483 alkaline protease comprising one or more of the following mutations: S3T, V41, R99G, R99A, R99S, S154D, S1564E, A188P, V193M, V199I, L211D and L211.
  • a liquid enzymatic detergent composition is prepared by mixing together at room temperature about 13 wt % C10-C13 linear alkylbenzene-sulfonic acid, about 5 wt % alkylpolyglycoside(C12-C14), about 10 wt % of a C13 alcohol polyethoxylate having 7 EO units, about 6 wt % lauric acid, about 7 wt % of oleic acid, about 5 wt % triethanolamine, about 5 wt % propanediol 1,2, about 2 wt sodium hydroxide, about 1 wt % citric acid, about 7 wt % ethanol, about 1 wt % citric acid about 7 wt % ethanol, about 1-hydroxyethane-1,1-diphosphonic acid and the remainder being water.
  • the pH of the resulting solution is 8.1 (measured as a 10% aqueous solution).
  • a sufficient amount of the subtilisin protease comprising one or more of the following mutations: R99G, R99A, R99S, R99E, L211D, L211E, S154D and S154E, is added to yield a composition having about 0.5 wt % of liquid protease concentrate (1250 HPE/g).
  • a similar composition as described in example 13 is prepared except the protease is replaced by a mutated Bacillus lentus DSM 5483 alkaline protease comprising one or more of the following mutations: S3T V41, R99G, R99A, R99S, S154D, S154E, A188P, V193M, V199I, L211D and L211E.

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